(Supported in part by NSF MCB 9420772 to B. McEwen). The goal of this TRD is to perfect high-pressure freezing for three different types of samples 1) tissue culture monolayers that will subsequently be freeze-substituted and flat-embedded in plastic; 2) suspensions that will also be freeze-substituted and embedded; and 3) suspensions that will be cut as frozen-hydrated sections. We are using mitotic PtK1 cells to develop techniques for monolayer applications. Our intent is to obtain an optimal morphological description of kinetochores in PtK1 cells through out mitosis. Ultimately we will combine high-pressure freezing/ freeze-substitution with immuno-labeling and mutant transfection studies to build a molecular model of kinetochore function. For high-pressure freezing PtK1 cells are seeded on to small aclar disks (approximate diameter = 1.0 mm) that fit into the high-pressure freezing hats. The disks are cut from sheets of aclar plastic using a specially designed punch. After two days growth the disks are scanned for mitotic cells growing on the top surface. Disks are transferred to high-pressure freezing hats as soon as they are selected. The volume of the hat is filled with either 1-hexadecene or 10% ficoll in tissue culture media and the sample immediately put into the high pressure freezer. After freezing the samples are kept under liquid nitrogen while transferring to the freeze-substitution media. The later is either 2% osmium tetroxide in acetone or 1% glutaraldehyde plus 0.1% tannic acid in acetone followed by 1% osmium tetroxide plus 0.1% uranyl acetate in acetone. Freeze-substitution is accompanied by a staged increase in temperature from -90 0C to room temperature over a 1-2 day period. Generally all of the aclar disks are recovered after freeze-substitution but varying numbers of cells are inevitably ripped from the surface of the disk. As a result, at least half of the disks no longer have mitotic cells when scanned via phase-contrast LM. We are currently investigating the suitability of applying low levels of poly-lysine to help anchor cells to the aclar disks. Disks that still have mitotic cells after freeze-substitution are placed on glass coverslips with the cells away from the glass. Epon is added and after the plastic has cured the glass coverslip is removed by etching with hydrofluoric acid. Areas of the flat embedment containing disks are cut out and remounted on an epon peg. The aclar disk is then gently cut away from the block leaving the cells embedded at the surface of the plastic where they can be readily sectioned. Initially we found good freezing in all but the mitotic cells on a disk that was frozen in the old-style specimen hats. Specimens in the new-style hats were consistently poorly frozen, presumably because the new hats were not designed for, and imperfectly fit into, our old-style freezing rods. Subsequently we have found consistently good freezing for non-mitotic cells using the old-style hats and we are currently examining some mitotic cells that appear to be well frozen. In the future the problem of freezing hats will have to be addressed, presumably by purchasing new specimen rods, but the ten old-style hats we have are sufficient for the developing the methodology. We are also experimenting with different freeze-substitution protocols. We started with a protocol from the Martin M|ller's laboratory and have subsequently switched to a protocol provided by Mary Morphew of the Boulder (CO) Resource. We are also in contact with Kent McDonald of the Univ. California (Berkeley) and h ave received helpful hints concerning orientation of the aclar disks for efficient embedding. Rat liver was high-pressure frozen and freeze-substituted using the Muller protocol, with good results. The membrane contrast was somewhat better than in some blocks we received from Muller. We found a marked increase in the intracristal space with time the liver pieces spent in buffer before freezing. We are making tomographic reconstructions of three time periods, as a model of mitochondrial degeneration. In the best-preserved material, however, the mitochondria were very dense, and the membranes were hard to see. We next plan to prepare mitochondria with the Morphew freeze-substitution protocol which includes tannic acid and glutaraldehyde, followed by osmium and uranyl acetate. Skills were developed for cutting thin and semi-thick (0.25lm) frozen-hydrated sections, uisng the high-pressure frozen rat-liver material, following suggestions from Muller's papers. A method of attaching the small pieces of material to the microtome chuck using special low-temperature glue was found to be satisfactory. Thin sections could be collected on bare grids, pressed in place by a metal bar, while the thicker sections had to be placed in folding grids. As reported in the literature, the sections fragment during handling of the grids, and we did not have on hand the recommended special fine-mesh grids for these first trials. Nevertheless section fragments examined by cryo-EM in the IVEM seemed to have useable contrast, but reliable assessment of the quality of the frozen-hydrated sections awaits repeats with the new grids.